The HU Protein Is Important for Apicoplast Genome Maintenance and Inheritance in Toxoplasma gondii

Abstract

The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritance remain largely unstudied. Here we genetically characterize a nucleus-encoded homolog to the bacterial HU protein in Toxoplasma gondii. In bacteria, HU is a DNA-binding structural protein with fundamental roles in transcription, replication initiation, and DNA repair. Immunofluorescence assays reveal that in T. gondii this protein localizes to the apicoplast. We have found that the HU protein from Toxoplasma can successfully complement bacterial ⌬hupA mutants, supporting a similar function. We were able to construct a genetic knockout of HU in Toxoplasma. This ⌬hu mutant is barely viable and exhibits significant growth retardation. Upon further analysis of the mutant phenotype, we find that this mutant has a dramatically reduced apicoplast genome copy number and, furthermore, suffers defects in the segregation of the apicoplast organelle. Our findings not only show that the HU protein is important for Toxoplasma cell biology but also demonstrate the importance of the apicoplast genome in the biogenesis of the organelle.

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins

“…The 100 ng of the resulting template was used for qPCR reaction using SYBR Green Mix (Bio‐Rad) and primers Apg‐qPCR‐F/R for apicoplast and UPRT‐qPCR‐F/R for nuclear genomes. Copy number control was performed using specific plasmids as described in .…”

Host cell metabolism contributes to delayed-death kinetics of apicoplast inhibitors in Toxoplasma gondii

“…We selected three inhibitors that are well-documented to cause delayed-death in T. gondii and have strong evidence for their target in the apicoplast: actinonin (membrane metalloprotease FtsH1), clindamycin (ribosome), and ciprofloxacin (DNA gyrase) (5, 10, 12, 13, 20). During treatment with these compounds, the apicoplast had been previously observed by microscopy of T. gondii RH parasites expressing an apicoplast-targeted ferredoxin NADP+ reductase fused to red fluorescence protein (FNR-RFP) (12, 18, 21–24). In these experiments, treatment with apicoplast inhibitors leads to vacuoles with some parasites missing FNR-RFP fluorescence (10, 12, 18).…”

“…During treatment with these compounds, the apicoplast had been previously observed by microscopy of T. gondii RH parasites expressing an apicoplast-targeted ferredoxin NADP+ reductase fused to red fluorescence protein (FNR-RFP) (12, 18, 21–24). In these experiments, treatment with apicoplast inhibitors leads to vacuoles with some parasites missing FNR-RFP fluorescence (10, 12, 18). While these studies suggested that the inhibitors lead to disruption of apicoplast biogenesis, it was unclear how exactly FNR-RFP fluorescence corresponded to apicoplast presence.…”

“…While these studies suggested that the inhibitors lead to disruption of apicoplast biogenesis, it was unclear how exactly FNR-RFP fluorescence corresponded to apicoplast presence. Furthermore, since these studies used microscopy, they could only count apicoplasts in vacuoles containing <8 parasites, corresponding to no more than 3 replications out of >6 total replications during the lytic cycle (10, 12, 18). To clarify these initial observations, we sought to develop a quantitative method to monitor apicoplast loss through a full lytic cycle.…”

“…How parasites are able to compensate for this loss during the first lytic cycle remains poorly understood. Of note, growth kinetics resembling delayed death have also been observed for inhibitors that block apicoplast metabolic function and genetic disruption of proteins required for apicoplast biogenesis or metabolism, suggesting that inhibiting production of essential apicoplast metabolites may be the common perturbation leading to delayed death in T. gondii (5, 15–18).…”